Copper is an essential, yet toxic, micronutrient. Copper serves as a cofactor of enzymes that are vital for normal growth and development. Genetic disorders in copper metabolism, copper-implicated degenerative diseases, and nutritional copper deficiency in various organisms provide striking evidence that homeostatic copper metabolism is an essential physiological process. In the context of these clinical correlations, characterization of the copper transport system represents an important biomedical problem. Ctrl, a highly conserved family of integral membrane protein in eukaryotes, has been identified. While roles of Ctrl in copper metabolism have just begun to be elucidated, the mechanisms of action and regulation of Ctrl which is critical for optimal copper acquisition remain to be determined. Physical and functional interactions between Ctrl and other components involved in copper metabolism are not fully understood. The long-term goals of this proposal are to understand the molecular mechanisms of homeostatic copper transport across the plasma membrane and characterization of the pathways whereby organisms acquire optimal levels of copper. This application focuses on elucidation of the mechanisms of action and regulation of Ctrl copper transporter. Our preliminary data show that the structure, expression levels, subcellular localization and activity of Ctrl are delicately controlled in a copper-dependent manner. Our central hypothesis is that several layers of post-translational regulation of Ctrl maintain optimal cellular copper acquisition. A multi- disciplinary approach combining physiology, biochemistry, cell biology, and genetics will be employed to pursue the following specific aims: 1) We will charaterize molecuar events in the Ctrl multimeric complex that are coupled with copper transport. 2) We will elucidate the roles of the Ctrl amino terminal domain in copper transport. 3) We will define the physiological significance, structural determinants, and signaling involved in copper-induced red ox-sensitive multimerization of Ctrl. Given that Cu acquisition is a central step in Cu metabolism, defining the function, mode of action and regulation of Ctrl could lead to better insights into Cu homeostasis. Considering the fact that Wilson disease, Menkes disease, Alzheimer's disease, and other severe degenerative disorders are linked to defects in copper homeostasis, studies on the molecular mechanisms of copper acquisition would ultimately advance our ability to combat copper-related pathologies.